Polymer adjuvants for innate and cellular based vaccination

在商业疫苗生产的早期，同一疫苗不同批次的效力之间的显著差异被正确地归因于反应容器的污染。然而，对清洁的更严格的关注似乎降低了疫苗的有效性，这表明污染物实际上增强了免疫原性。这些污染物被描述为“佐剂”；它们刺激免疫系统并增强对疫苗的反应，而不具有任何固有的抗原性。他们通过创造一个非特异性的促炎环境来做到这一点，在这种环境中，呈现特定的抗原导致疫苗反应要有效得多。经验表明，佐剂对有效疫苗的发展起到了至关重要的作用，许多佐剂现在被广泛使用，包括油、铝盐和病毒体。疫苗接种领域的重要性与日俱增，从1977年成功根除天花(估计挽救了3000万人的生命)到迫切需要艾滋病毒、疟疾、结核病和癌症的预防性(理想的治疗性)疫苗。新的佐剂将对新疫苗的开发做出重要贡献，因为一些迄今最有效的佐剂(如弗氏佐剂)不能生产成cGMP，不适合临床使用。解决这一不足的一种方法是设计和生产适合cGMP生产的有效和特异的合成分子佐剂。我们将合成能够与其受体结合并激活程序化细胞因子反应的聚合物-TLR配体结合物。利用可控自由基聚合法合成多价反应性聚合物，可得到分子量分布较窄的聚合物。亲水性单体如聚乙二醇甲基丙烯酸酯或N，(2-羟丙基甲基丙烯酰胺)将与活化的含酯单体或含叠氮化物的单体共聚，以进行后续的改性。为了确定材料诱导免疫反应的最佳能力，将考察聚合物结构的三个参数：第一，聚合物的相对分子质量和多分散性；第二，每个聚合物中TLR配体的数量，最后是间隔基的长度和性质。所有这些参数都将被平行调整，对于了解材料的特性如何改变免疫反应非常重要。最初将使用报告细胞来评估生物活性，这些细胞在任何TLR配体的刺激下被激活。报告蛋白被表达，并反映细胞的激活水平，因此是刺激的指标。其次，将通过测量激活后释放的细胞因子的相对量来评估配体-聚合物结合物的比活性，以确定这些材料是刺激基于细胞的免疫反应还是基于抗体的免疫反应。

In the early days of commercial vaccine manufacture, significant variation between the effectiveness of different batches of the same vaccine were correctly ascribed to contamination of the reaction vessels. However, more scrupulous attention to cleanliness seemed to reduce the effectiveness of the vaccines, suggesting the contaminants actually enhanced immunogenicity. These contaminants were described as 'adjuvants'; agents which stimulate the immune system and increase the response to a vaccine, without having any intrinsic antigenic effect. They do this by creating a non-specific pro-inflammatory environment, where presentation of specific antigens leading to a vaccine response is much more efficient. Experience shows that adjuvants make a crucial contribution to the development of effective vaccines, and many adjuvants are now in widespread use, including oils, aluminium salts and virosomes.The field of vaccination is of ever-increasing importance, ranging from successful eradication of Smallpox in 1977 (estimated to have saved 30 m lives) through to the pressing need for prophylactic (and ideally therapeutic) vaccines for HIV, malaria, TB and cancer. New adjuvants will make an important contribution to the development of new vaccines, as some of the most effective adjuvants to date (such as Freund's adjuvants) cannot be manufactured to cGMP and are not suitable for clinical use. One way to address this shortfall is to design and produce potent and specific synthetic molecular adjuvants which are suitable for cGMP manufacture.We will synthesize polymer-TLR ligands conjugates which are capable of binding their receptor and activate a programmed cytokine response. Multivalent reactive polymers will be synthesized using controlled radical polymerization to obtain polymers which have a narrow molecular weight distribution. Hydrophilic monomers such as polyethylene glycol methacrylate, or N,(2-hydroxypropylmethacrylamide) will be co-polymerized with either an activated ester bearing monomer or an azide bearing monomer for subsequent modification. Three parameters of polymer structure will be examined in order to determine optimum capacity of the materials to induce an immune response: Firstly the molecular weight and polydispersity of the polymer; secondly the number of TLR ligands per polymer and finally the spacer length and properties. All of these parameters will be adjusted in parallel and are important in understanding how features of the material alter the immune response.Biological activity will be initially assessed using reporter cells which are activated upon stimulation of any TLR ligands. A reporter protein is expressed and reflects the level of activation of the cells and thus an indicator of stimulation. Secondly, the specific activity of the ligand-polymer conjugates will be assessed to determine whether the materials stimulate either a cell based or antibody based immune response by measuring the relative quantities of the cytokines released following activation.